Core staff are available to assist with experimental design and to answer questions related to metagenomic approaches. Arrangements for a meeting to discuss new or existing projects can be made by sending an email to Nathan Bivens, core director.

Assistance with grant proposals and letters of support is also available by contacting the director.

Prior to sample submission and services performed, a project must be initiated with the core director and involves the following steps.

1. Contact the core director to discuss project details. Several parameters are needed to quote a project and establish the scope of the work to be performed.
   • Number of samples
   • Organism/sample source
   • Number of sequence reads per sample to be generated.
   • MO code for project charges.
   
   
2. The core director will prepare a Project Authorization Form (PAF) outlining the services. The form must be signed and returned before services are started.

3. A Project Information Form should be completed and submitted by email prior to sample submission.

The MUGTC provides a targeted amplicon approach for evaluating and understanding complex bacterial communities. Our pipeline provides a streamlined process for the analysis of 96 samples simutaneously.

The Bioinformatics and Analytics Core can provide an analysis of the data using the Qiime pipeline, http://qiime.org/. This will provide graphical and tabular summaries of the taxa and OTUs identified and their relative proportional representations.

Standard amplicon primer sets:

Target Region	Primer Name	Primer Sequence	Amplicon Length (nt)*
16S V4	U515F	5'-GTGCCAGCMGCCGCGGTAA-3'	430
	806R	5'-GGACTACHVGGGTWTCTAAT-3'
ITS1	ITS1-30F	5'-GTCCCTGCCCTTTGTACACA-3'	535
	ITS1-217R	5'-TTTCGCTGCGTTCTTCATCG-3'

Custom amplicon primer design:

PCR Round	Primer Name	Primer Sequence
Round 1*	Forward Primer	5'-acactctttccctacacgacgctcttccgatct NNNNNNNNNNN-3'
	Reverse Primer	5'-gtgactggagttcagacgtgtgctcttccgatct NNNNNNNNNNN-3'
* First round amplification to be performed by the researcher.
Round 2	i5 Index Primer	5'-aatgatacggcgaccaccgagatctacac[i5]acactctttccctacacgacgctcttccgatct-3'
	i7 Index Primer	5'-caagcagaagacggcatacgagat[i7]gtgactggagttcagacgtgtgctcttccgatct-3'

Design considerations:

1. Lowercase letters denote Illumina adapter sequences necessary for binding to the flow cell
2. Underlined lowercase are binding sites for the Illumina sequencing primers
3. [i5] and [i7] refer to dual indexing sites; 8 bases per index
4. Sequence of uppercase "N's" are region specific portion of round 1 primers

General Considerations:

1. Submit cells in Eppendorf DNA Lo Bind Tubes, 0.2ml (Cat. No. 022431048)
2. The preferred 96-well plate for submitting targeted amplicon samples is from MedSupply Partners (877-633-7779, http://www.medsupplypartners.com/); catalog # 15-3590. The core recommends the use of adhesive sealing sheets from Thermo Scientific (cat. #AB-0558).

Library Type	Quantity Required	Concentration	Recommended Buffer	Special Instructions
DNA (genomic)	1000 - 2000 ng	100 - 200 ng/µl	EB buffer; 10 mM Tris-Cl (pH 8.5)	An image of 100 ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity.
Total RNA Stranded (rRNA depletion)	500 - 1000 ng	50 - 200 ng/µl	nuclease-free water	Provide sample source and extraction information on methods used.
Target amplicon (16S & ITS)	210 ng	3.5 ng/µl volume = 60 µl	EB buffer; 10 mM Tris-Cl (pH 8.5)	We strongly recommend DNA be quantified by Qubit assay.
Custom amplicon (2-step PCR)	60 ng	1 ng/µl volume = 60 µl	EB buffer; 10 mM Tris-Cl (pH 8.5)	We strongly recommend DNA be quantified by Qubit assay.

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Library Type	Sequence Depth*	Sequence Type	Sequence Read	Cycle Number
16S V4 Amplicon	100,000 reads/sample	Paired-end, unique, dual indexing	Read 1 i7 Index i5 Index Read 2	250 cycles 8 cycles 8 cycles 250 cycles
ITS Amplicon	100,000 reads/sample	Paired-end, unique, dual indexing	Read 1 i7 Index i5 Index Read 2	300 cycles 8 cycles 8 cycles 300 cycles
Metatranscriptome	20 million reads/sample	Paired-end, unique, dual indexing	Read 1 i7 Index i5 Index Read 2	100 cycles 8 cycles 8 cycles 100 cycles

* minimum read count recommended; increased sequencing depth may be desired for some studies

Library Prep Type	Fee
Genomic Library Preparation Total RNA Library Preparation (rRNA depletion) 16S Amplicon Generation (96 samples) ITS Amplicon Generation (96 samples) Custom Amplicon Generation	$125 $200 $370 $370 Inquire

NovaSeq 6000	Fee
NovaSeq SP - PE50 NovaSeq SP - PE100 NovaSeq SP - PE150 NovaSeq SP - PE250 NovaSeq S1 - PE50 NovaSeq S1 - PE100 NovaSeq S1 - PE150 NovaSeq S2 - PE50 NovaSeq S2 - PE100 NovaSeq S2 - PE150 NovaSeq S4 - PE100 NovaSeq S4 - PE100 (lane) NovaSeq S4 - PE150 NovaSeq S4 - PE150 (lane)	$3,848 $4,457 $4,6731 $6,044 $5,661 $6,755 $7,193 $9,383 $11,299 $11,955 $15,594 $3,939 $17,209 $4,343
MiSeq	Fee
MiSeq PE75 MiSeq PE150 MiSeq PE250 MiSeq PE300 MiSeq PE150 - Nano MiSeq PE250 - Nano	$1,488 $1,647 $1,796 $2,201 $785 $852